This R21 application is in response to RFA-AI-05-042 on Innate Immunity to Category B pathogens of which Enterocytozoon bieneusi, a Microsporidium previously classified as protozoa. Of the 14 species affecting human health E. bieneusi is clinically the most significant emerging enteric pathogen that infects the gastrointestinal tract of most mammalian species, and is the major cause of chronic diarrhea, wasting and cholangitis in patients with HIV/AIDS, malnourished children and those receiving immunosuppressive therapy. The technical difficulties that were associated with the lack of in vitro laboratory propagation methods as well as limited sources of spores, has contributed to the very slow progress on understanding the biology, pathogenesis and protective immune responses against this emerging pathogen. These have to a large extent been overcome recently by our group, which provides the impetus to this application. The long term goal of this application is to enhance our understanding of the mechanisms involved in E. bieneusi protective immunity. This information is critical for the development of effective immunotherapeutic approaches that may help resolve otherwise a fatal infection in immunodeficient individuals. Our preliminary data indicate that IFN-gamma is an important component in providing initial resistance to E. bieneusi infection. An investigation into the molecular basis of cellular activation by E bieneusi, including a characterization of the innate immune receptors that initiate this initial resistance is unknown. The goals of this application are to identify the specific cellular receptors and adaptor proteins that are responsible for initiating innate immunity during E. bieneusi infection, and to determine which IFN-gamma-dependent components are regulated by the host immune system during infection of epithelial cells. The role of TLRs in innate immunity and the IFN-gamma regulated genes that are essential in the context of this infection, will be investigated. The specific aims are: [unreadable] 1. To examine the expression of E. bieneusi-specific IFN-gamma regulated genes that may be involved in innate immunity to infection. [unreadable] 2. To determine the role of Toll-like receptors (TLR)/MyD88 signaling pathway, in the induction of IFN-gamma,[unreadable] in response to E. bieneusi infection. Elucidation of the mechanistic basis of regulation of the innate immunity will lead to a better understanding of resistance to E. bieneusi infection. Moreover, innate immunity significantly affects the generation of acquired immunity to many infections. Thus, the proposed studies will form a foundation on which to build further studies to examine how regulation of innate immunity impacts acquired immunity to this emerging infection. [unreadable] [unreadable] [unreadable]